A Protein-Coding Circular RNA That Regulates DNA Damage Response in BCR::ABL-Positive Leukemias

Circular RNAs (circRNAs) are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. CircRNAs are covalently joined and characterized by the perturbed arrangement of exons known as back-splicing. Initially regarded as transcriptional byproducts, circRNAs have been shown to regulate mRNA translation by acting as microRNA sponges, and recent studies have revealed their roles in transcription, translation, and various cellular functions. In cancer, circRNAs can function as oncogenes or tumor suppressors, and their stability makes them potential biomarkers for disease. In acute leukemias, circRNAs generated from recurrent chromosomal translocations contribute to leukemogenesis. Here, we investigate the role of circPCMTD1 in chronic myeloid leukemia (CML) in the blast crisis (BC).

Functional studies using LNA-modified, RNase H-recruiting oligonucleotides (gapmers) targeting circPCMTD1 demonstrated a significant decrease in proliferation and a potent G2/M cell cycle blockade in CML-BC cell lines (K-562 & LAMA-84), both harboring the t(9;22)(q34;q11.2). Quantitative real-time PCR confirmed the specificity of circPCMTD1 depletion without affecting the linear PCMTD1 transcript. CircPCMTD1 knockdown (KD) reduced the viability of leukemic blasts, indicating its essential role in cell survival.

RNA sequencing after circPCMTD1-KD in K-562 cells identified approximately 150 differentially expressed genes involved in cell cycle control, nuclear organization, and transcriptional regulation, such as SMARCA4, MACM, PCLAF, and SASH1. Gene Set Enrichment analysis highlighted rRNA processing and DNA replication-dependent chromatin function to be notably affected by circPCMTD1 depletion. CyTOF-based cell cycle analysis validated the G2/M blockade. Increased γH2AX levels indicated aberrant DNA damage response, confirmed by western blotting and intracellular flow cytometry. In addition, circPCMTD1-KD led to an increase in the phosphorylation of the CHK1, RPA32, ATR, ATM, and DNA-PK proteins. DNA fiber assays and comet assays further confirmed reduced DNA replication capacity and increased double-stranded DNA breaks upon circPCMTD1 depletion. Taken together, these data underscore the aberrant DNA damage response and the significant increase in genotoxic stress that is triggered by circPCMTD1 depletion.

We performed targeted circPCMTD1 profiling in CML patients in the chronic, accelerated, and blast crisis phase and found an increased abundance of circPCMTD1 in advanced disease stages, indicating a potential role of higher circPCMTD1 expression in disease progression. In vitro experiments with patient blasts showed that circPCMTD1-KD increased γH2AX levels specifically in BCR::ABL-positive samples. In vivo, targeting circPCMTD1 in mice engrafted with BCR::ABL-positive blasts prolonged survival significantly, with no notable toxicities observed.

Mechanistically, circPCMTD1 was enriched in the cytoplasm and associated with ribosomes. Polysome profiling suggested its protein-coding capacity, and we identified a cryptic open reading frame within circPCMTD1. Using custom antibodies, we detected a circPCMTD1-derived peptide (~30 KD) localized mainly in the nucleus. Immunoprecipitation followed by mass spectrometry revealed that the peptide interacted with BLM, TOP3A, and RMI1 proteins of the BTR complex. CircPCMTD1 knockdown reduced BTR complex formation. Knockdown of these proteins individually reduced leukemic blast viability, but concomitant depletion mimicked the G2/M blockade seen with circPCMTD1 depletion. Furthermore, treatment with Dasatinib, a tyrosine kinase inhibitor, decreased circPCMTD1-derived peptide levels without affecting the expression levels of the circPCMTD1 transcript and reduced BTR complex formation, linking BCR::ABL activity to circPCMTD1 function.

In summary, we identify circPCMTD1 as a crucial regulator in BCR::ABL-positive leukemias, affecting DNA damage response, proliferation, and cell cycle progression. Our findings highlight circPCMTD1 as a potential therapeutic target in myeloid malignancies with t(9;22). Future studies should explore the therapeutic implications of targeting circPCMTD1 in combination with existing treatments, potentially offering a novel approach to managing drug resistance and improving outcomes in CML patients.